The strain of West Nile virus now endemic in the continental United States is more virulent than the virus originally isolated in Africa and is classified as a category B priority pathogen by the NIAID. In only six years, it has spread throughout the continental United States, resulting in high morbidity and mortality. Last year, of the 2,539 cases reported to the CDC, nearly half of the patents had neuroinvasive symptoms. There is currently no vaccine or drug that combats the West Nile virus infection and only symptomatic treatment is available. Our ultimate goal is to develop a prophylactic or therapeutic treatment for this disease. To accomplish this, we will develop methods for the identification of compounds that inhibit the West Nile viral NS3 protease. The protease is a key enzyme in viral maturation and inhibition of flavivirus protease has been shown to dramatically reduce viral replication. Structure based drug design will provide a rapid path to potent and virus specific protease inhibitors. The crystal structure of this protease in the presence and absence of known peptide based inhibitors will be determined in an effort to characterize the active site. We will virtually screen the active site with a chemical library. Careful attention will be made to the composition of the library to ensure enrichment with diverse, pharmacologically active, lead-like compounds. A biochemical assay will be developed that has a wide range of sensitivity that can be used in the identification of weak binding initial hits from our virtual screen as well as to measure small changes in inhibition as we optimize these hits in future research. Compounds that are selected in the virtual screen and are active in the biochemical assay will be cocrystallized with the protease, giving us a clear picture of the compound's fit into the active site. This will position us to rapidly identify the best candidates from the active compounds and subsequent optimization in phase 2 research. [unreadable] [unreadable] [unreadable]